2 Alternative Carbohydrate Reserves Used in the Daily Cycle of Crassulacean Acid Metabolism
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2 Alternative Carbohydrate Reserves Used in the Daily Cycle of Crassulacean Acid Metabolism CC. BLACK, J.-Q. CHEN, ILL,. DOONG, M.N. AN(;EI.OV, illld S.J.S. SUN<; 2.1 Introduction Each day a massive interlocked biochemical cycle occurs in the Greta 1 issllcs of crassulacean acid metabolism plants. The function of this interlocked l,t::ee, in its simplest context, is to furnish most ofthe CO, for (‘AM plant photosynthesis. In addition, this diel (24 h) cycle produces the primary identifying marks of a ( ‘AM tissue through two ancillary cycles. One cycle involves a noctllrnal acitlific~ittiou and its loss the next day, while the second concerns the depletion of a carl>ohy- drate reserve at night and its replenishment the next day. Formally IIeniamiu Heyne (18 1s) is credited with writing, nearly two centuries ago, about I~IC “at,id as sorrel” taste of a succulent green plant at dawn and the “bland taste” causttcl by acidity loss later in the day. In fact, the exact origins of I hese observaliorls art‘ lost in antiquity, but certainly are referred to in Roman and Biblical wrilings The circumstantial cause of the acidity was postulated to be an organic acid aboul a century ago and the bland taste later was associated with starch; but ~hrse idr;ls were not plainly coupled together in theory nor quantitatively studietl lentil the late 1940s. Then, with the discovery of major portions of intermediary mr~al~- olism and the advent of additional quantitative biochemical procetlrlres. 111e nature of the daily reciprocal relation between the acid and the bland tastt* was recognized and measured quantitatively. The acid taste is caused principally Ily malic acid. while the bland taste is caused by deacidification plus the rocipl OCYII synthesis of a bland tasting carbohydrate, e.g. a Pc’lys~lcchal.ide such i\s stalch. Other daily ancillary cycles, e.g. CO, and O2 exchange, stomatal fuuctious, au internal pool of CO,, etc. also exist as integral parts of CAM (Klupe and I‘illg 1978; Ting and Gibbs 1982; Winter 1985). The focus of this work, however. is OII the type of carbohydrate and how each is metabolized in certain <‘AM ~LIIIIS when it functions as the daily carbon reservoir to provide the ~~l~osl~hoc,rl~~l- pyruvate (PEP) for nocturnal CO, fixation and organic acid synthesis. ‘l‘c~iay alternative pathways of intermediary carbohydrate metabolisnl in I~lli\l~ts arc known (Sung et al. 1988) and alternative carbohydrate reserves are recogniit*d ill specific CAM species (Black et al. 1982) which can be either a I”)lys~tcc.li:lrid~, OI a ---- Deparhnent of Biochemislry, Lire Sciences Huilding, The I Jniversity (II (iron $1. AIII~IN ( i/t 30602, USA 32 CC. Black et al. neutral hexose sugar. Unfortunately, these disparate bodies of knowledge have not been fully integrated with CAM. Therefore, in this unified presentation our aims are (1) to divide CAM plants into two metabolic groups, (2) to document the use of different carbohydrate reserves by each group, (3) to integrate the unique biochemical reactions within each group into characteristic sets of metabolic path- ways, thereby depicting two metabolic sequences of carbon cycling in CAM, and (4) to compare the bioenergetics and other features of these two metabolic groups. 2.2 The Division of CAM Plants into Two Metabolic Groups The division of CAM plants into distinct metabolic groups was proposed when two types of C,-acid decarboxylases were found in CAM plants. Malic enzyme was first assayed in certain CAM plants in the mid 1950s but it was not widely studied nor considered to act as a decarboxylase until the late 1960s only several years after the 1965 discovery of C, photosynthesis (Black 1973). Thus in 1973 when a second decarboxylase, PEP carboxykinase, was found to be very active in specific CAM species, it was clear that two large groups of CAM plants existed (Dittrich et al. 1973). The details of these separate metabolic conversions were not clear, but, over the intervening two decades, new information about carbon metabolism has been found in higher plants and distinct metabolic variations have been discovered in other CAM plants. Table 2.1 is a condensed presentation of earlier work on the taxonomic distribution of some carbon metabolism enzymes among CAM plants. In the initial work classifying CAM plants into two groups, the activities of PEP carboxykinase and pyruvate, Pi dikinase were used as the basis of division (Dittrich et al. 1973). Pyruvate, Pi dikinase shows a taxonomic pattern comple- mentary to PEP carboxykinase (Table 2.1). But some enzymes were active in all CAM plants, however, with elevated activity in one group. For example, both types of malic enzyme tended to be expressed more in plants without PEP carboxykinase, whereas the pyrophosphate-dependent phosphofructokinase (PFK) tended to be more active in plants containing a highly active PEP carboxykinase. Even with these limitations, data as in Table 2.1 gave a reasonable basis for the division. 2.3 The Use of Soluble Sugars Versus Polysaccharides as a Carbohydrate Reserve Starch was identified in early CAM work as the likely source of carbon for the nocturnal synthesis of organic acids (Bennet-Clark 1933; Wolf 1937). For several decades, research on CAM tissues was dominated by efforts to understand the night CO, fixation, 0, metabolism, the respiratory quotient, and other daily gas exchange traits (Kluge and Ting 1978; Edwards and Walker 1983) (see also Chap. 1). Some clever ideas were published to “explain” the unusual gas exchange Alternative Carbohydrate Reserves Used in the Daily Cycle 33 Table2.1. Taxonomic distribution of enzymes amongst CAM.plants separated into two groups based on PEP carboxykinase activity Family (number of species) PEPCK” NAD-ME” NADP-ME” PPDK” PP,-PFK” pmol mg-’ Chl h-’ Active PEP carboxykinase Asclepiadaceae(3) 1733440 10-90 2-44 nilb Bromeliaceae( 12) 193-999 16-140 6-91 nil 25- 107 Euphorbiaceae(2) 625-830 167-232 - nil Asphodelaceae(2) 178-480 29-62 - nil - Vitaceae( 1) 597 106 - - - Little PEP carboxykinase Agavaceae(3) NDb 43-785 144 40-50 - Aizoaceae(2) ND 195 - - Asteraceae(2) ND 50 - 80 Cactaceae(3) ND 47-920 131 30-200 2’ Crassulaceae(5) ND 140-385 46-212 90- 240 2-8’ Orchidaceae(3) ND 60-190 151-217 30-200 - Dracaenaceae(2) ND 50-127 120 70 - “PEPCK: PEP carboxykinase; NAD- or NADP-ME: malic enzyme; PPDK: Pyruvate, phos- phate dikinase; PP,-PFK: pyrophosphate-dependent phosphofructokinase. The level of detec- tion via the assays employed was < 5 umol mg- ’ Chl h-r. Data collected about 1983 from Kluge and Osmond (1971); Dittrich et al. (1973); Sugiyama and Laetsch (1975); Black (1976); Dittrich (1976); Holtum and Osmond (1981); Black et al. (1982); and Carnal and Black (1983). bNot detectable. A dash indicates no assay reported. Nil indicates a detectable activity but usually less than 1 umol mg-’ Chl h-‘. ‘Activity in umol mg-’ protein h-t. traits of whole CAM plant tissues. Nevertheless, there was little meaningful understanding of how CAM functioned biochemically. Hence, while the general phenomenon of an acid taste in succulent plants was recognized for centuries, no integrated model existed until the late 1940s. Then in a remarkable effort to unify the current knowledge about CAM gas exchange, M. Thomas presented a scheme [initially in the third edition of his textbook (Thomas 1947) and subsequently in a research paper (Thomas 1949)] for the interconversion of the carbohydrate and “vegetable acids” as given below: Carbohydrate -co2 I t 4 I Products of glycolysis Vegetable acids I t +co2 This simple loop-like scheme was given without further comment. Even so, in distilled essence, it is the model that guides the research on how CAM functions even today! These relationships were strongly supported in the series of quantitative 34 C.C. Black et al. studies on total acids and carbohydrates by H.B. Vickery and coworkers (Pucher et al. 1949; Vickery 1954) who demonstrated that these die1 synthesis and degrada- tion processes were the reverse of each other, occurring in a reciprocal fashion .each day. These quantitative relationships between starch and malic acid quickly allow- ed them to fit their data into a similar scheme. For example with Bryophyllum calycinum ( = KalanchoP pinnata), starch loss and acid accumulation balanced (Pucher et al. 1949). Indeed, in K. pinnata, starch loss at night was 50% higher than required for acid synthesis (Sutton 1975a, b). Even though putatively the carbohydrate was starch, in some cases, starch could not account for the amount of acids synthesized. For example, in K. tubijloru and K. daigremontiuna, starch accounted for only two-thirds of the carbon (Sutton 1975a, b), and in Opuntia uurantiuca less than 40% of the carbon was derived from starch (Whiting et al. 1979). In each of these plants however, the glucan (low-molecular-weight poly- mers of glucose) pool change was sufficient to account for the remaining carbon and these authors concluded that soluble sugar pools did not contribute carbon for PEP synthesis. But in other studies, on the effects of changing environments, Vickery (1954) could not totally account for the malate carbon from starch even in B. calycinum (K. pinnutu), which underscores the strong responses of CAM to environmental conditions (Kluge and Ting 1978). It is somewhat ironic that, simultaneously with this excellent work done in Connecticut on balancing acidity with a polysaccharide, the first work with soluble sugars as a potential carbon reserve appeared in the literature with pineapple from Hawaii (Sideris et al. 1948). The exact pathway of carbohydrate metabolism in pineapple leaves had not been understood because substantial amounts of sugars were depleted each night concurrent with malate synthesis.